A broad variety of therapeutic polynucleotides have been delivered to cells by receptor-mediated endocytosis. When linked to a target cell-specific ligand, the polynucleotide is cointernalized by the target cell along with the ligand. Once inside the cell, the polynucleotide is released in functional form, for example, as an expressible gene or as an antisense construct which inhibits expression of an endogenous gene. In the case of plasmid DNA, the plasmid is maintained in the target cell as a non-replicating episome without integrating into the cell's genome (Wilson et al. (1992) J. Biol. Chem 267(16):11483-11489).
The first report of receptor-mediated gene transfer resulting in detectable expression of the gene was by Wu et al. (1987) J. Biol. Chem. 262: 4429-4432. Wu et al. developed a novel system for delivering polynucleotides to cells based on their work involving the introduction of genes into hepatocytes via the asialoglycoprotein receptor (see also, Wu et al. (1988) J. Biol. Chem. 263: 14621-14624; Wu et al. (1989) J. Biol. Chem. 264: 16985-16987; Wu et al. (1991) J. Biol. Chem. 266: 14338-14342; U.S Pat. No. 5,166,320).
The system developed by Wu et al. employs a soluble polynucleotide-carrier complex made up of a gene or other polynucleotide electrostatically linked to a bifunctional carrier molecule. The carrier molecule, comprised of a polycation-ligand conjugate, serves the dual function of linking the gene (via the polycation moiety, e.g., polylysine) and binding to the target cell (via the ligand, e.g., an asialoglycoprotein), resulting in internalization of the carrier molecule by the cell.
To form the molecular complex, Wu et al. linked the gene to the carrier in a step-down dialysis from a high salt solution of approximately 2.0-3.0 M NaCl down to approximately 0.15 M, thereby slowly complexing the negatively charged DNA and the positively charged polycation-ligand carrier. Wu et al. recognized that the method used to form the complex must result in a structure which (a) is soluble in solution so that it can easily pass through physiological barriers when administered in vivo to reach target cells or tissues, (b) is stable extracellularly so that the polynucleotide remains linked to the carrier, and (c) releases the polynucleotide in functional form under intracellular conditions.
The polynucleotide delivery system developed by Wu et al. has since been used by several others to deliver a broad variety of genes to selected cells (see e.g., Chen et al. (1994) Human Gene Therapy 5:429-435; Ferkol et al. (1993) FASEB 7: 1081-1091; Midoux et al. (1993) Nucleic Acids Research 21(4):871-878; Martinez-Fong (1994) Hepatology 20(6):1602-1608; Plank et al. (1992) Bioconjugate Chem. 3:533-539; Wagner et al. (1990) PNAS 87:3410-3414; Chen et al. (1994) FEBS Letters 338:167-169; Ferkol et al. (1993) J. Clin. Invest. 92:2394-2400; Rojanasakul et al. (1994) Pharmaceutical Res. 11(12):1731-1736; and Ross et al. (1995) Human Gene Therapy 6:31-40). However, during this time, some variations on the method of forming the DNA complex have been made in an effort to increase the level and duration of expression obtained from the targeted gene.
For example, Cotten et al. (1990) PNAS 87: 4033-4037 and Wagner et al. (1991) PNAS 88: 4255-4259 formed polynucleotide-carrier complexes containing plasmid DNA linked to a transferrin-polycation carrier molecule. In contrast to the step-down dialysis method employed by Wu et al., Cotten et al. and Wagner et al. directly mixed the transferrinpolycation conjugate and the plasmid DNA, with and without additional free polycation, at a concentration of 0.15 M NaCl. Wagner et al. recognized that the polycation not only served to link the DNA, but also functioned to condense the DNA into small toroid structures of approximately 80-100 nanometers in diameter, facilitating its uptake by cells.
Perales et al. (1994) PNAS 91: 4086-4090 also modified Wu's system for forming polynucleotide-carrier complexes, with the goal of condensing the DNA to form highly compacted complexes which are easily taken into endosomes. Specifically, Perales et al. linked a plasmid encoding human factor IX to galactosylated polylysine by titration with increasing concentrations of NaCl, resulting in complexes of approximately 10-12 nanometers in diameter as measured by electron microscopy (see also, WO 95/25809).
In contrast to the step-down dialysis method of Wu et al. and the direct mixing method of Wagner et al., Perales et al. slowly added the carrier dropwise to a solution of the plasmid, both at 0.7 M NaCl, over the course of 30 minutes to one hour. This step resulted in a turbid, aggregated solution which was then slowly titrated with 3 .mu.l aliquots (allowing at least 30 seconds between the addition of each new aliquot) of 5 M NaCl until a clear, unaggregated solution was obtained at a "critical" concentration of NaCl, somewhere between 0.97 M NaCl and 1.03 M NaCl. Further 2 .mu.l aliquots of 5 M NaCl were then gradually added if a precipitate formed. The authors observed that when increasing the ionic strength of the mixture above the critical salt concentration, the DNA complexes assumed a non-functional rod-like conformation of increased diameter. They therefore conclude that the concentration of NaCl must be kept at or near the critical range, generally between 0.5 M and 1.5 M (roughly 3.5 to 10 times physiological levels) (see WO 95/25809 at page 35, lines 10-11).
There remains a need for a more practical method of forming high performance polynucleotide-carrier complexes which produce persistent and high levels of gene expression when delivered to cells both in vitro and in vivo.